During the proposed extension of this R37, we will continue to pursue our overall hypothesis that differences in the expression of the ADH genes affect the metabolism of alcohols, the risk for alcoholism, and the consequences of alcohol consumption on different tissues. We will locate regulatory sequences in the ADH genes and their flanking regions, and study the effects of genetic variation intrre-form of different natural haplotypes on gene expression. We will examine the effects of epigenetic modifications (DNA methylation, histone acetylation) on gene expression, also in the context of individual variations in the key modified sequences (CpG sequences that are subject to methylation) or in the enzymes that affect these modifications. The specific aims include 1. Identify and analyze the regulatory effects of cis-acting elements upstream-of the ADH genes on gene expression in the context of natural haplotypes. We will usetransient transfections, protein-DNA binding assays, identification of new genetic variations by resequencing. The effects of different haplotypes will be examined. 2. Identify and analyze regulatory elements and epigenetic regulation throughout the ADHgene cluster using the genomic DNA arrays we are developing. We hypothesize that distant elements affect regulation of the ADH genes. We will detect sites of hyperacetylated histones, sites at which transcription factors bind, DNase I hypersensitive sites, and sites of DNA methylation across the entire ADH gene cluster. These sites will be examined after over-expression and inhibition of transcription factors (Aim 3) and under conditions including ethanol exposure. We will then characterize-individual sequence differences in these regulatory sites, and study the effects of those differences on ADH gene expression as in Aim 1. 3. Determine which transcription factors bind to key regulatory sites and analyze their effects on gene expression. We will determine which factors bind to key sequences and examine both primary and secondary effects of overexpression and inhibition of specific factors using reporter assays, RT-PCR of endogenous genes, and microarrays. These specific aims form a coordinated.project that addresses the regulation otADH gene expression and individual differences in gene expression. We know of no other group examining the regulation of ADH genes in this comprehensive detail or with a focus on individual genetic differences. Such an examination is critical to our understanding of the effects of these genes on risk for alcoholism and alcohol-related organ damage